Preparation of albumin using PEG and EDTA

ABSTRACT

Methods for the large scale preparation of clinical albumin are disclosed. One method includes dilution of plasma with a NaCl solution containing disodium ethylene dinitrilo tetraacetate and a stabilizer such as sodium caprylate. The resulting solution is then subjected to heating and cooling steps, followed by the addition of polyethylene glycol to precipitate impurities, with albumin remaining in the supernatant. Isoelectric precipitation is then employed to recover the desired albumin product, which is devoid of the albumin dimer. In an alternative method, ethanol is employed as the precipitating agent rather than polyethylene glycol.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to the preparation of albumin. Moreparticularly, the present invention is directed to a method in whichlarge-scale preparation of clinical albumin is facilitated. The methodhas the advantages of giving a significantly higher yield than prior artprocedures and of requiring less than one-half the processing time ofsuch known procedures as the Cohn procedure.

Previous large-scale procedures for the preparation of albumin haveincluded those described by Cohn et al. in J. Amer. Chem. Soc. 68,459-475 (1946) and J. Amer. Chem. Soc. 72, 465-474 (1950). Otherprocedures for the preparation of albumin have included that describedby W. Schneider et al., Blut. 30: 121-134 (1975). Such prior artprocedures, in general, have not resulted in satisfactory yields andhave required long processing periods in order to prepare the albumin.The present invention for the preparation of albumin has the advantagesof providing a significantly higher yield than the Cohn et al. procedureand of requiring less than one-half the processing time of the Cohn etal. procedure.

The present method for the preparation of albumin includes the steps of:(1) dilution of plasma in liquid form with an equal volume of an aqueousNaCl solution containing: (a) EDTA (diosodium ethylene dinitrilotetraacetate) to break the iron-transferrin complex and possibly othermetallo proteins; and (b) a stabilizer such as sodium caprylate: (2)heating the plasma-containing solution at approximately 60° C. for about11/2 hours at a pH of 6.2±0.1; (3) cooling the solution to about 10° C.;(4) precipitation of impurities of the heated solution with PEG 4000(polyethylene glycol) at approximately 18-20% by weight concentration,leaving the albumin in the supernatant; (5) isoelectric precipitation ofalbumin from the PEG supernatant at a pH of about 4.6; and (6)recovering the resultant albumin which is devoid of the albumin dimer.

In the literature there is a description by Inman et al., Vox Sang. 6:34-52 (1961) of a large-scale method for the purification of humantransferrin. In the literature method, the use of EDTA and ethanol atlow temperature is described for decreasing the solubility oftransferrin during the purification of this protein. The method of thepresent invention includes the use of EDTA and heating, on the otherhand, in order to reduce the stability of transferrin and other metallicproteins, thus effecting conformational changes which facilitate theirprecipitation by 20% PEG, leaving a highly purified albumin in thesupernatant.

In an alternative method of the present invention, the steps include:(a) adjusting the pH of plasma in liquid form to about 6.7; (b) heatingthe plasma at a temperature of approximately 60° C. for about 11/2hours; (c) adjusting the pH of the plasma to about 5.7; (d)precipitating impurities from the plasma by the addition of ethanol inan amount sufficient to give a final concentration of about 40 to 44% byvolume in the plasma, along with cooling of the plasma to about -5° C.,with the albumin remaining in the supernatant; and (e) adjusting the pHof the supernatant to about 4.8 to precipitate albumin from thesupernatant.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the method of the present invention for preparing clinical albumin ona large scale, the method was carried out in a series of steps which aredescribed hereinafter as Example I, with Example I being presented inthe form of a flow chart.

The plasma employed should be in liquid form at a temperature such as,for example, about 5° C. The salinity of the NaCl solution should beselected so as to approximate the salinity of the plasma itself.

The amount of EDTA employed may vary, with an amount as low as about 2mMhaving been employed in Example I with good results.

EXAMPLE I

    ______________________________________                                        Flow Chart for Preparation of Albumin                                          ##STR1##                                                                     ______________________________________                                    

Any suitable stabilizer other than sodium caprylate may be employed, solong as the stabilizer functions to stabilize the albumin in solution.Under the conditions of Example I, from about 4mM to about 8mM ofstabilizer are generally employed.

The method of the present invention results in increased yields of thedesired product, in an amount of about 28.5 g/liter, for example, whenthe method is carried out at a pilot plant scale at MDPH (MichiganDepartment of Public Health) of 55 liters. In accordance with thepresent invention, the use of EDTA has been found to render several ofthe metallo proteins unstable and precipitable during the heatingprocess. The present method has the additional feature of providing analbumin product which is devoid of the albumin dimer, the latter beinginvariably found in the product prepared according to currentlyavailable procedures.

In an alternative method of the present invention, preparation ofclinical albumin on a large scale was carried out in a series of stepswhich are described hereinafter as Example II, with Example II beingpresented in the form of a flow chart.

EXAMPLE II ##STR2##

As shown in Example II, the alternative method includes the steps of:(a) adjusting the pH of plasma in liquid form to about 6.7; (b) heatingthe plasma at a temperature of approximately 60° C. for about 11/2hours; (c) adjusting the pH of the plasma to about 5.7; (d)precipitating impurities from the plasma by the addition of ethanol inan amount sufficient to give a final concentration of about 40 to 44% byvolume in the plasma, along with cooling of the plasma to about -5° C.,with the albumin remaining in the supernatant; and (e) adjusting the pHof the supernatant to about 4.8 to precipitate albumin from thesupernatant. The pH may be adjusted throughout the above steps by theuse of, for example, a 0.8 M acetate buffer. As further shown in ExampleII, the albumin obtained by the precipitation may be subjected tofurther steps of reconstitution, lyophilization, filtration andpasteurization to obtain the final product, with the pH being adjustedas indicated by a material such as a dilute NaOH solution.

The methods of Example I and II have been evaluated on the 55-60 literscale and there has been obtained a yield of albumin of 28.1 grams/literby the method of Example II. These methods compare favorably with theCohn procedure which produced albumin in the amount of from 22 to 23grams/liter.

It is thought that the invention and many of its attendant advantageswill be understood from the foregoing description, and it will beapparent that various changes may be made in the methods as describedherein without departing from the spirit and scope of the invention orsacrificing its material advantages, the forms hereinbefore describedbeing merely preferred embodiments thereof.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method for thepreparation of albumin comprising:(a) diluting plasma in liquid formwith an equal volume of a NaCl solution containing disodium ethylenedinitrilo tetraacetate and a stabilizer; (b) adjusting the pH of theplasma solution resulting from step (a) to about 6.2; (c) heating theplasma solution from step (b) at a temperature of approximately 60° C.for about 11/2 hours; (d) cooling the plasma solution to about 10° C.;(e) precipitating impurities from the solution with polyethylene glycolat a concentration of about 18-20% with the albumin remaining in thesupernatant; (f) isoelectrically precipitating albumin from saidsupernatant at a pH of about 4.6; and (g) recovering the albuminproduct.
 2. The method of claim 1 wherein said stabilizer is sodiumcaprylate.